5-fluoro-2&#39;-deoxyuridine derivative, processes for preparing same and antitumor composition containing the same

ABSTRACT

A 5-fluoro-2&#39;-deoxyuridine derivative expressed by the following formula ##STR1## wherein R 1  and R 2  are the same or different from each other, each representing an alkyl group of 1 to 18 carbon atoms having a carboxyl group as a substituent, or an alkyl group of 9 to 14 carbon atoms, or its pharmacologically acceptable salt as the active ingredient. 
     Said 5-fluoro-2&#39;-deoxyuridine derivative displays a high-level antitumor effect in low doses and shows outstanding safety.

TECHNICAL FIELD

The present invention relates to an antitumor composition. Moreparticularly, this invention relates to an antitumor compositioncomprising, as its effective ingredient, a 5-floro-2'-deoxyuridinederivative which shows strong antitumor activity even in low doses,provides a very wide margin of safety and displays the highly sustainedreleasing effect in vivo for 5-fluoro-2'-deoxyuridine.

BACKGROUND ART

5-Fluorouracil is known as the compound having antitumor activity and tospeak of its mode of action, it takes in vivo a metabolic pathway to5-fluorouridinemonophosphate, then to 5-fluorouridinediphosphate, andfurther to 5-fluoro-2'-deoxyuridinemonophosphate, which inhibitsthymidylate synthetase activity, thus providing the antitumor effect.Said 5-fluoro-2'-deoxyuridinemonophosphate is metabolizable further into5-fluoro-2'-deoxyuridine.

The last-mentioned 5-fluoro-2'-deoxyuridine, one of said metabolites, isalso known as the compound having an antitumor activity. However, it hasbeen reported that, though 5-fluoro-2'-deoxyuridine shows a strongantitumor activity in vitro, it fails to exercise enough antitumoreffect in experiments conducted in vivo on tumor-bearing animals (CancerResearch, 19, 494 (1959); Proc. Soc. Exp. Biol., N.Y., 97, 470 (1958);Proc. Soc. Exp. Biol., N.Y., 104, 127 (1960); Ann., N.Y. Acad. Sci., 76,576 (1958)).

This is attributed to the fact that, in in vivo, the half-life period of5-fluoro-2'-deoxyuridine is extremely short, so there is not enough timefor 5-fluoro-2'-deoxyuridine to be in contact with the tumor cells(Cancer Research, 32, 1045 (1972); Clin. Pharmacol. Ther., 5, 581(1964); Cancer Research, 38, 3479 (1978); Bull. Cancer (Paris), 66, 67(1979); Bull. Cancer (Paris), 66, 75 (1979); Europ. J. Cancer, 16, 1087(1980)).

Studies have hitherto been made by many specialists on5-fluoro-2'-deoxyuridine in order to overcome the disadvantage mentionedabove.

For example, 3-acyl-5-fluoro-2'-deoxyuridines (Japanese PatentApplication Laid-open No. 163586/'79) and3-acyl-3',5'-di-0-acetyl-5-fluoro-2'-deoxyuridines (Japanese PatentApplication Laid-open Nos. 113797/'81 and 113795/'81) are known. Thesecompounds, however, have not had their antitumor effect improvedsufficiently enough and there is still a drawback in terms of theirsafety (therapeutic index).

It has also been reported that 3',5'-diacyl-5-fluoro-2'-deoxyuridineswhich have their 3'- and 5'-positions acylated with alkanoyl groups haveantitumor activity (Biochemical Pharmacology, 14, 1605 (1965)).According to this report, 3',5'-diacyl-5-fluoro-2'-deoxyuridines havetheir antitumor activity tested always at doses ranging from 10 to 40mg/kg/day, i.e., doses at which its parental compound5-fluoro-2'-deoxyuridine showed antitumor activity. However, it has notyet been ascertained whether it has a significantly high level ofantitumor activity and therapeutic index comparable to that of5-fluoro-2'-deoxyuridine.

It is reported in the Cancer Chemother. Pharmacol., (1981) 6: 19˜23,that, of the 3',5'-diacyl-5-fluoro-2'-deoxyuridine family whose 3'- and5'-positions are acylated with an alkanoyl group,3',5'-diacyl-5-fluoro-2'-deoxyuridine which has its 3'- and 5'-positionsacylated with an acetyl (C₂), propanoyl (C₃), butyryl (C₄), hexanoyl(C₆), or palmitoyl (C₁₆) group exhibits an antitumor effect at a lowerdose as compared with 5-fluoro-2'-deoxyuridine. With regard to thetherapeutic index, however, these 3',5'-diacyl-5-fluoro-2'-deoxyuridinesshow an improvement by only 2 to 3 times that of5-fluoro-2'-deoxyuridine.

DISCLOSURE OF THE INVENTION

The present invention provides an antitumor composition which comprisesa 5-fluoro-2'-deoxyuridine derivative expressed by the following formula(I) ##STR2## wherein R₁ and R₂ are the same or different from eachother, each representing an alkyl group of 1 to 18 carbon atoms having acarboxyl group as a substituent, or an alkyl group of 9 to 14 carbonatoms, or their pharmacologically acceptable salts as the effectiveingredient.

The 5-fluoro-2'-deoxyuridine derivative proposed by the presentinvention is a compound which shows strong antitimor activity even inlow doses, provides a markedly improved therapeutic effect (therapeuticindex), and displays the highly sustained releasing effect in vivo for5-fluoro-2'-deoxyuridine. The compound, which comprises the5-fluoro-2'-deoxyuridine derivatives of this invention in which R¹ andR² are an alkyl group of 1 to 18 carbon atoms having a carboxyl group asa substituent, is a novel compound.

BEST MODE OF CARRYING OUT THE INVENTION

R¹ and R² of the 5-fluoro-2'-deoxyuridine derivative expressed by theaforementioned formula (I) of the present invention are the same ordifferent from each other and represent an alkyl group of 1 to 18 carbonatoms having a carboxyl group as a substituent, or an alkyl group of 9to 14 carbon atoms. As the alkyl group of 1 to 18 carbon atoms having acarboxyl group as substituent, there are straight chained or branchedalkyl groups. Said carboxyl group may be substituted for a terminalcarbon atom or any carbon atom other than a terminal carbon atom in thealkyl group. It is desirable for an alkyl group to have one carboxylgroup substituted. As the alkyl group of 1 to 18 carbon atoms having acarboxyl group as the substituent, carboxymethyl, 2-carboxyethyl,3-carboxypropyl, 4-carboxybutyl, 5-carboxypentyl, 6-carboxyhexyl,7-carboxyheptyl, 8-carboxyoctyl, 9-carboxynonyl, 10-carboxydecyl,11-carboxyundecyl, 12-carboxydodecyl, 13-carboxytridecyl,14-carboxytetradecyl, 15 -carboxypentadecyl, 16-carboxyhexadecyl,17-carboxyheptadecyl, 18-carboxyoctaceyl, 3-carboxy-3-methylbutyl,2-carboxydecyl, 2-carboxydodecyl, and 2-carboxytetradecyl, for instance,may be mentioned.

As the alkyl group of 9 to 14 carbon atoms, nonyl, decyl, undecyl,dodecyl, tridecyl, and tetradecyl, for instance, may be mentioned andnonyl, undecyl, and tridecyl are especially desirable.

The 5-fluoro-2'-deoxyuridine derivative of this invention can be used inthe form of pharmacologically permissible salt. As the pharmacologicallypermissible salt, salts of carboxyl group may be usable in case where R₁and R₂ are alkyl group of 1 to 18 carbon atoms having a carboxyl groupas the substituent. As the salt of this type, such salts of alkalimetals as sodium salt and potassium salt; such divalent or trivalentmetal salts as calcium salt, magnesium salt, and aluminum salt; and suchorganic salts as ammonium salt, tetramethylammonium salt,monomethylammonium salt, dimethylammonium salt, trimethylammonium salt,morpholine salt, and piperidinium salt, for instance, may be mentioned.

As another pharmacologically permissible salt, acid addition salts withan acid added to the nitrogen atom at the 3-position may also be usable.As such an acid, such inorganic acids as hydrochloric acid, hydrobromicacid, sulfuric acid and phosphoric acid; such organic carboxylic acidsas acetic acid, propionic acid, citric acid, succinic acid, tartaricacid, and maleic acid; and such organic sulfonic acids asmethane-sulfonic acid, ethanesulfonic acid, benzenesulfonic acid, andp-toluenesulfonic acid, for instance, may be mentioned.

To give examples of the 5-fluoro-2'-deoxyuridine derivative, thefollowing compounds may be mentioned:

3',5'-dimalonyl-5-fluoro-2'-deoxyuridine,

3',5'-disuccinyl-5-fluoro-2'-deoxyuridine,

3',5'-diglutaryl-5-fluoro-2'-deoxyuridine,

3',5'-diadipoyl-5-fluoro-2'-deoxyuridine,

3',5'-dipymeryl-5-fluoro-2'-deoxyuridine,

3',5'-disuberyl-5-fluoro-2'-deoxyuridine,

3',5'-disubesyl-5-fluoro-2'-deoxyuridine,

3',5'-didecanoyl-5-fluoro-2'-deoxyuridine,

3',5'-didodecanoyl-5-fluoro-2'-deoxyuridine,

3',5'-ditetradecanoyl-5-fluoro-2'-deoxyuridine.

The 5-fluoro-2'-deoxyuridine derivative of formula (I) preparedaccording to the present invention shows an excellent effect ofprolonging the life span of a tumor bearing mouse upon administration inlow dose ranging from 1/10 to 1/130 of the dose required for5-fluoro-2'-deoxyuridine to show the similar degree of antitumor effecton prolonging the life span of the mouse bearing L1210 mouse leukemiacells. It has also been made clear that its therapeutic index, whichshows the therapeutic advantage quantitatively, and is determined bydividing the dose required to obtain the maximum survival time of theL1210 bearing mouse (TLS max) by the dose required to increase thesurvival time by 30% (ILS₃₀), is 5 to 20 times higher than that of itsparent compound, 5-fluoro-2'-deoxyuridine.

It has further been clarified that the 5-fluoro-2'-deoxyuridinederivative of this invention shows sustained release of5-fluoro-2'-deoxyuridine by enzymatic reaction which takes place in thesystem of porcine liver esterase, etc. in vitro.

The foregoing facts substantiate a claim that the compound of thisinvention has a high antitumor effect because the5-fluoro-2'-deoxyuridine derivative prepared according to this inventioneffects a sustained release of 5-fluoro-2'-deoxyuridine whose half lifein vivo is very short and keeps it in contact with the tumor cells for along period of time in the living body.

As mentioned above, the 5-fluoro-2'-deoxyuridine derivative of thepresent invention has an excellent antitumor effect, and accordingly anantitumor drug which comprises the 5-fluoro-2'-deoxyuridine derivativeexpressed by the aforementioned formula (I) as the effective ingredientcan be provided according to the present invention.

The 5-fluoro-2'-deoxyuridine derivative of the present invention can beadministered orally or parenterally such as subcutaneously,intramuscularly, intravenously, percutaneously, and rectally. Of thesemethods, oral and intravenous administrations are most advisable. As thedosage form for oral administration, tablets, pills, granules, powders,liquid preparations, suspensions, emulsions, liposome preparations, andcapsules may be mentioned.

The preparation of tablets can be achieved according to any ordinarymethods of forming tablets by use of such excipients as lactose, starch,crystalline cellulose, and hydroxypropyl cellulose; such binders ascarboxymethyl cellulose, methyl cellulose, polyvinyl pyrrolidone, andsodium alginate; and such disintegrators as calcium carboxymethylcellulose and starch. Pills, powders, granules, etc. can also be formedaccording to the ordinary method by use of such excepients, etc. asmentioned above. Emulsions and suspensions can be prepared according tothe ordinary method by use of such glycerin esters as tricapyrtin,triacetin, and trilaurin; such vegetable oil a coconuts oil andfractionated coconuts oil; and such alcohols as ethanol. In preparingcapsules, hard gelatin capsules may be filled with granule or powder andsoft gelatin capsules may be filled with a liquid preparation.

As the form of dosage to be administered subcutaneously, intramuscularlyor intravenously, there are injections prepared in the form of aqueousor nonaqueous solutions, suspensions, emulsions, and liposomepreparations. In preparing nonaqueous solutions and suspensions,propylene glcyol, polyethylene glycol, olive oil, and ethyl oleate, forinstance, are used and antiseptics and stabilizers are further added, ifnecessary. In the case of preparing aqueous solutions, such surfactantsas polyoxyethylene hardened castor oil, polyoxyethylene sorbitanmonolaurate, polyoxyethylene hardened castor oil, polysorbate 20,polysorbate 80, and polyoxyethylene sorbitan monooleate may be added asthe solubilizing agent. As the lipid to be used for preparing emulsionsand liposome preparations, vegetable oils, lecithin, phosphatidylethanolamine, phosphatidyl inositol, phosphatidyl serine, sphingomyelin,and phosphatidic acid cholesterol may be mentioned. As the stabilizer tobe used for preparing emulsions and liposome preparations, dextran,albumin, vinyl polymer, monionic surfactant, gelatin, and hydroxyethylstarch may be used. Injections are usually sterilized by subjecting themto filtration through a bacterial filter or by treating them with abactericide.

The dosage form for percutaneous administration includes, for instance,ointments and creams. Ointments are prepared according to ordinarymethods by use of such fatty oils as castor oil and olive oil, andvaseline. Creams are prepared according to ordinary methods by use offatty oil or such emulsifying agents as ethylene glycol and monosorbitanfatty acid esters.

For rectal administration, gelatin soft capsules and suppositoriesprepared by use of cacao butter are used.

The dose of the 5-fluoro-2'-deoxyuridine derivative of this inventionvaries depending upon the age and sex of a patient, the condition andnature of a disease, and the dosage form; however, the usual dose is0.005 to 9 mg/kg/day, preferably 0.01 to 4 mg/kg/day.

The amount of the 5-fluoro-2'-deoxyuridine derivative to be contained inthe antitumor composition of the present invention is determined inconsideration of said dose. For example, the usual dose in the form of atablets, capsules, injections, etc. shall contain 0.1 to 180 mg,preferably 0.2 to 80 mg, of the 5-fluoro-2'-deoxyuridine derivative.

The 5-fluoro-2'-deoxyuridine derivative of this invention can be givento a patient in dosage of more than one kind of them in combination.

In case where R₁ and R₂ of the 5-fluoro-2'-deoxyuridine derivativesexpressed by formula (I) of this invention are alkyl group of 9 to 14carbon atoms, such compounds can all be made synthetically according toany publicly known methods as shown, for instance, in the BiochemicalPharmacology, 14, 1605 (1965).

To give an example, they can be synthesized according to the ordinarymethod wherein 5-fluoro-2'-deoxyuridine is made to react with itscorresponding acid halide or acid anhydride in the presence of suchorganic base as pyridine and trialkylamine.

The 5-fluoro-2'-deoxyuridine derivatives of this invention whose R₁ andR₂ are alkyl groups of 9, 11, and 13 carbon atoms respectively aredisclosed in the Biochemical Pharmacology, 14, 1605 (1965) and arepublicly known compounds.

In the case where R₁ and R₂ of the 5-fluoro-2'-deoxyuridine derivativeexpressed by formula (I) of this invention are respectively alkyl groupof 1 to 18 carbon atoms having a carboxyl group as the substituent,namely the 5-fluoro-2'-deoxyuridine derivative expressed by thefollowing formula (I') ##STR3## wherein R₁ ' and R₂ ' are the same ordifferent from each other, each representing an alkyl group of 1 to 18carbon atoms having a carboxyl group as a substituent, or theirpharmaceutically acceptable salts, they are novel compounds.

These compounds can be synthesized according to the following methods.

(1) In the case where R₁ ' and R₂ ' are the same:

In the case where R₁ ' and R₂ ' are the same, the5-fluoro-2'-deoxyuridine expressed by the following formula (I'-a)##STR4## wherein R₁ ' and R₂ " are the same, representing an alkyl groupof 1 to 18 carbon atoms having a carboxyl group as a substituent, can beprepared by allowing 5-fluoro-2'-deoxyuridine to react with carboxylicacid expressed by the following formula (II)

    R.sub.1 'COOH                                              (II)

wherein R₁ ' is as defined hereinabove, or its reactive derivative inthe presence of base.

As the reactive derivative of carboxylic acid of formula (II), suchcorresponding acid halides as acid chloride and acid bromide, acidanhydride, mixed acid anhydride, activated ester, and activated acidamide may be mentioned. Such acid halides as acid chloride and acidbromide are especially preferable of all.

As the base to be used in the reaction between carboxylic acid or itsreactive derivative and 5-fluoro-2'-deoxyuridine, such organic bases astrimethylamine, triethylamine, tributylamine, pyridine,N-methylmorpholine, 2,6-lutidine, and N,N-dimethylaminopyridine; andsuch inorganic bases as alkali acetates and alkali carbonates may bementioned. Of these bases, such organic bases as pyridine andtriethylamine ae preferable. As the reaction solvent, nonpolar solventsincluding such ethers as ethyl ether, tetrahydrofuran, and dioxane; suchhydrocarbon halogenides as methylene chloride and carbon tetrachloride;and such aromatic hydrocarbons as benzene and toluene may be amongdesirable solvents. To speak of the amounts of the respective species tobe used in the reaction, it is necessary to use more than two molarequivalents of carboxylic acid expressed by formula (II) or its relativederivative against 5-fluoro-2'-deoxyuridine. The base is also used inmore than two molar equivalents. It is advisable to carry out on thereaction with cooling by use of ice in the initial stage and,thereafter, to carry out the reaction at room temperature. The reactiontimes varies depending upon the kind and quantity of the compounds to beused in the reaction, however, it is usually in the range of 1 to 5hours.

Though the 5-fluoro-2'-deoxyuridine derivative of this invention is thusobtained, it may further be subjected to the salt-forming reaction inorder to obtain its pharmacologically permissible salt, if necessary.The salf-forming reaction is effected according to the ordinary method.To adduce an example, when any of sodium hydroxide, potassium hydroxide,sodium carbonate, ammonia, trimethylamine, monoethanolamine, andmorpholine is made to react with the 5-fluoro-2'-deoxyuridine derivativefor neutralization according to the ordinary method, the salt of acarboxyl group in the molecule of the 5-fluoro-2'-deoxyuridinederivative is obtained.

An acid addition salt of the 5-fluoro-2'-deoxyuridine derivative can beobtained by bringing the 5-fluoro-2'-deoxyuridine into contact with anyof inorganic acids, organic carboxylic acids, and organic sulfonic acidsin an organic solvent.

The isolation and purification of the desired compound after thereaction can be carried out according to the ordinary methods such asrecrystallization, thin-layer chromatography, and column chromatography.

(2) In the case where R₁ ' and R₂ ' are different from each other:

In the case where R₁ ' and R₂ ' are different from each other, the5-fluoro-2'-deoxyuridine derivative expressed by the following formula(I'-b) ##STR5## wherein R₁ ' and R₂ "' are different from each other,each representing an alkyl group of 1 to 18 carbon atoms having acarboxyl group as a substituent, can be synthesized by making thecompound expressed by the following formula (III) ##STR6## wherein Rrepresents a protective group react with carboxylic acid expressed bythe following formula (II)

    R.sub.1 'COOH                                              (II)

wherein R₁ ' is as defined hereinabove, or its reactive derivative inthe presence of base. After the release of the protective group from thereaction product, the reaction product is allowed to react withcarboxylic acid expressed by the following formula (IV)

    R.sub.2 "'COOH                                             (IV)

wherein R₂ "' is as defined hereinabove or its reactive derivative inthe presence of base, followed by the salt-forming reaction, if requiredso.

In the compound expressed by the aforementioned formula (III) to be usedin the present invention, R indicates a protecting group. As theprotecting group, the protecting groups like triphenylmethyl group andtriphenylmethoxyacetyl group which have high steric hindranceperformance may be mentioned. The compound of the aformentioned formula(III) can be prepared according to the ordinary method.

The reaction between the compound of formula (III) and carboxylic acidof formula (II) or its reactive derivative can be conducted according tothe same method as mentioned above with an exception of making themreact in amounts equimolar with each other.

The releasing of the protective group can be effected under the ordinaryhydrolysis conditions in acidic or alkaline solutions; more particularlyunder the acidic conditions in aqueous solution of acetic acid orhydrochloric acid or under the alkaline conditions in ammoniac methanolsolution.

After the release of the protective group, the reaction of theintermediate reaction product with carboxylic acid of formula (IV) orits reactive derivative can be carried out after the same method asmentioned above with an exception of using carboxylic acid of formula(IV) or its reactive derivative in equimolar amounts. The salt-formingreaction can also be effected in the same way as mentioned above.

The foregoing are the methods by which the 5-fluoro-2'-deoxyuridinederivative or its pharmacologically acceptable salt is prepared.

The following Examples illustrate the invention in detail.

EXAMPLE 1

Synthesis of 3',5'-diadipoyl-5-fluoro-2'-deoxyuridine

To a solution of 250 mg (1.01 mmol) of 5-fluoro-2'-deoxyuridine in 10 mlof anhydrous pyridine was added 800 mg (4.37 mmol) of adipoyl chloridefor about 3 hours, and the mixture was stirred overnight at roomtemperature. The reaction mixture was then poured into 50 ml of ice-coldwater and stirred for 1 hour. To the reaction mixture was added 2N HClto adjust to pH 4.00, and the reaction product was extracted three timeswith 20 ml of ethyl acetate. Ethyl acetate was distilled away at roomtemperature under reduced pressure and the obtained crude product wasdissolved in chloroform. The solution was purified by columnchromatography on silica gel. The chloroform-ethanol (95:5 to 90:10)eluants were collected and concentrated to yield3',5'-diadipoyl-5-fluoro-2'-deoxyuridine. The yield was 40%.

UV(λmax): 209 nm, 268 nm

NMR(δ_(CDCl).sbsb.3^(TMS) --D₃ COD): 1.5-1.8 (m, 8H), 2.1-2.5 (m, 10H),4.2-4.4 (m, 3H), 5.1-5.3 (m, 1H), 6.3 (t, 1H), 7.9 (d, 1H, J=6.5 Hz).

m.p.: 44°-45° C.

EXAMPLE 2

Synthesis of 3',5'-diglutaryl-5-fluoro-2'-deoxyuridine

To a solution of 220 mg (0.89 mmole) of 5-fluoro-2'-deoxyuridine in 3 mlof anhydrous pyridine was added 280 mg (2.46 mmole) of glutaricanhydride. The mixture was stirred overnight at room temperature and foranother 3 hours at 80° C. The reaction mixture was poured into 30 ml ofice-cold water and stirred for 1 hour. To the reaction mixture was added2N HCl to adjust to pH 4.00 and the reaction product was extracted threetimes with 15 ml of ethyl acetate. Ethyl acetate was distilled off atroom temperature under reduced pressure to obtain the crude product. Thecrude product was dissolved in chloroform and purified by columnchromatography on silica gel. The chloroform-ethanol (93:7 to 98:12)eluants were collected and concentrated to obtain oily3',5'-diglutaryl-5-fluoro-2'-deoxyuridine. The yield was 70%.

UV(λmax): 209 nm, 268 nm

NMR(δ_(D).sbsb.3_(COD) ^(TMS)): 1.7-2.2 (m, 4H), 2.2-2.7 (m, 10H),4.2-4.5 (m, 3H), 5.2-5.4 (m, 1H), 6.25 (t, 1H), 7.92 (d, 1H, J=6.5 Hz).

EXAMPLE 3

Synthesis of 3',5'-disuccinyl-5-fluoro-2'-deoxyuridine

To a solution of 500 mg (2.02 mmole) of 5-fluoro-2'-deoxyuridine in 6 mlof anhydrous pyridine was added 500 mg (5.00 mmole) of succine anhydrideat room temperature, and the mixtue was stirred overnight. The reactionmixture was poured into 60 ml of ice-cold water and stirred for 1 hour.To the mixture was added 2N HCl to adjust to pH 4.00, and the solutionwas extracted three times with 30 ml of ethyl acetate. The extracts wereconcentrated at room temperature under reduced pressure to obtain crudeproduct. The crude product was dissolved in chloroform and subjected tocolumn chromatography on silica gel. The fractions eluted at thechloroform-ethanol ratio of 90:10 to 85:15 were collected andconcentrated to give 3',5'-disuccinyl-5-fluoro-2'-deoxyuridine. Theyield was 80%.

UV(λmax): 209 nm, 268 nm

NMR(δ_(D).sbsb.3_(COD) ^(TMS)): 2.5-2.7 (m, 10H), 4.2-4.4 (m, 3H),5.2-5.4 (n, 1H), 6.25 (t, 1H), 7.92 (d, 1H, J=6.5 Hz).

m.p.: 116°-117° C.

EXAMPLE 4

Synthesis of 3',5'-bis-(β-carboxyundecanoyl)-5-fluoro-2'-deoxyuridine

A solution of 500 mg (2.02 mmol) of 5-fluoro-2'-deoxyuridine and 12.2 mg(0.1 mmol) of 4-dimethylaminopyridine in 15 ml of anhydrous pyridine wasprepared and 1,200 mg (5.0 mmole) of n-octyl succinic anhydride wasadded thereto at room temperature. The mixture was stirred at roomtemperature overnight. The reaction mixture was poured into 100 ml ofice water, and the mixture was stirred for 1 hour. To the mixture wasadded 2N HCl to adjust to pH 4.0, and the solution was extracted threetimes with 50 ml of chloroform. The extracts were concentrated at roomtemperature under reduced pressure to obtain crude product, and theproduct was dissolved in chloroform. The solution was passed through acolumn chromatography on silica gel and the eluates obtained at thechloroform-ethanol ratio ranging from 100:0 to 97:3 were collected andconcentrated to give3',5'-bis-(β-carboxyundecanoyl)-5-fluoro-2'-deoxyuridine. The yield was85%.

UV(λmax): 209 nm, 268 nm

NMR(δ_(CDCl).sbsb.3^(TMC)): 0.85 (t, 6H), 1.3 (s, 28H), 2.1-2.9 (m, 8H),4.2-4,4 (m, 3H), 5.1-5.2 (m, 1H), 6.15 (t, 1H), 7.9 (d, 1H, J=6.5 Hz).

EXAMPLE 5

Synthesis of 3',5'-bis-(β-carboxytridecanoyl)-5-fluoro-2'-deoxyuridine

A solution was first prepared by dissolving 500 mg (2.02 mmole) of5-fluoro-2'-deoxyuridine and 12.2 mg (0.1 mmole) of4-dimethylaminopyridine in 15 ml of anhydrous pyridine, and to thesolution was added 1,340 mg (5.0 mmole) of n-decyl succinic anhydride atroom temperature. The mixture was stirred at room temperature overnight.The reaction mixture was poured into 100 ml of ice water and stirred for1 hour. The reaction mixture was adjusted to pH 4.0 with 2N HCl andextracted three times with 50 ml of chloroform. The extracts wereconcentrated at room temperature under reduced pressure to obtain crudeproduct. The product was dissolved in chloroform and subjected to columnchromatography on silica gel. The fractions eluted at thechloroform-ethanol ratio ranging from 100:0 to 98.2 were collected andconcentrated to obtain3',5'-bis-(β-carboxytridecanoyl)-5-fluoro-2-deoxyuridine. The yield was80%.

UV(λ_(max) ^(EtOH)): 209 nm, 268 nm

NMR(δ_(CDCl).sbsb.3^(TMS)): 0.85 (t, 6H), 1.3 (s, 36H), 2.1-2.9 (m, 8H),4.2-4.4 (m, 3H), 5.1-5.2 (m, 1H), 6.15 (t, 1H), 7.9 (d, 1H, J=6.5 Hz).

EXAMPLE 6

Synthesis of 3',5'-bis-(β-carboxypentadecanoyl)-5-fluoro-2'-deoxyuridine

500 mg (2.02 mmole) of 5-fluoro-2'-deoxyuridine and 12.2 mg (0.1 mmole)of 4-dimethylaminopyridine were dissolved in 15 ml of anhydrous pyridineand 1,480 mg (5.0 mmole) of n-dodecyl succinic anhydride was added tothe solution at room temperature. The mixture was stirred overnight andthen adjusted to pH 4.0 with 2N HCl. The mixture was extracted threetimes with 50 ml of chloroform. The extracts were concentrated at roomtemperature under reduced pressure to obtain crude product. The productwas dissolved in chloroform and subjected to column chromatography onsilica gel. The eluates obtained at the chloroform-ethanol ratio rangingfrom 100:0 to 98:2 were collected and concentrated to give3',5'-bis-(β-carboxypentadecanoyl)-5-fluoro-2'-deoxyuridine in a 80%yield.

UV(λ_(max) ^(EtOH)): 209 nm, 268 nm

NMR(δ_(CDCl).sbsb.3^(TMS)): 0.85 (t, 6H), 1.3 (s, 44H), 2.1-2.9 (m, 8H),4.2-4.4 (m, 3H), 5.1-5.2 (n, 1H), 6.15 (t, 1H), 7.9 (d, 1H, J=6.5 Hz).

EXAMPLE 7

Synthesis of3',5'-bis-(3-carboxy-3-methylpentanoyl)-5-fluoro-2'-deoxyuridine

To a solution of 500 mg (2.02 mmole) of 5-fluoro-2'-deoxyuridine and12.2 mg (0.1 mmole) of 4-dimethylaminopyridine in 15 ml of anhydrouspyridine was added 710 mg (5.0 mmole) of 3,3-dimethylglutaric anhydrideat room temperature. The mixture was stirred at room temperatureovernight and then adjusted to pH 4.0 with 2N HCl. The mixture wasextracted three times with 50 ml of ethyl acetate. Ethyl acetate wasdistilled away at room temperature under reduced pressure to obtainedcrude product. The product was dissolved in chloroform. This solutionwas subjected to column chromatography on silica gel and the eluatesobtained at the chloroform-ethanol ratio of 100:1 to 95:5 were collectedand concentrated to give3',5'-bis-(3-carboxy-3-methylpentanoyl)-5-fluoro-2'-deoxyuridine. Theyield was 90%.

UV(λ_(max) ^(EtOH)): 209 nm, 268 nm

NMR(δ_(CDCl).sbsb.3^(TMS)): 1.12 (s, 12H), 1.9-2.5 (m, 10H), 4.2-4.4 (m,3H), 5.1-5.2 (m, 1H), 6.2 (t. 1H), 7.9 (d, 1H, J=6.5 Hz).

EXAMPLE 8

Antitumor activity of 5-fluoro-2'-deoxyuridine derivatives (inintraperitoneal administration)

Experiments were made with the 5-fluoro-2'-deoxyuridine derivatives ofthis invention in order to know its antitumor effect against mouseleukemia cells L1210 in comparison with its parent compound5-fluoro-2'-deoxyuridine and other known antitumor agents.

BDF₁ mice (male, 6-week-old, Ca 24 g, in groups of 5 mice), which wereabdominally implanted with 10⁵ cells of mouse leukemia L1210, which hadbeen collected after 7-day cultivation in other mice, were used in theexperiments.

The respective agents were given to the experimental miceintraperitoneally once a day for 5 consecutive days starting 24 hoursafter the implantation of tumor cells.

The antitumor effect of the respective agents were indicated by the rateof increase in the length of the survival period obtained from thegroups given the agents in contrast with the length of the survivalperiod of the control group (not given the agents).

The dose, which is required to increase the lifespan of the experimentalmice 30% as compared with the control group, is indicated by ILS₃₀ andthe dose, which is required to effect the maximum increase of lifespan(Max. ILS(%)), is indicated by ILS max. ILS max./ILS₃₀ is to representthe therapeutic index which indicates the safety of the agents.

The results of the experiments are as shown in Table 1.

                  TABLE 1                                                         ______________________________________                                                  Antitumor activity                                                              ILS.sub.30                                                                            ILS max         Therapeutic                                           (μmol ·                                                                   (μmol ·                                                                           index                                                 kg.sup.-1 ·                                                                  kg.sup.-1 ·                                                                   Max.   (ILS max./                                Compound    day)    day)     ILS (%)                                                                              ILS.sub.30)                               ______________________________________                                        Compounds of this                                                             invention:                                                                    3',5'-didecanoyl-5-                                                                       18      180      52     10.0                                      fluoro-2'-deoxy-                                                              uridine                                                                       3',5'-didodecanoyl-                                                                       1.0      45      62     45.0                                      5-fluoro-2'-deoxy-                                                            uridine                                                                       3',5'-ditetradeca-                                                                        --      4.5      61     --                                        noyl-5-fluoro-2'-                                                             deoxy-uridine                                                                 3',5'-diadipoyl-5-                                                                        --       20      58     20.0                                      fluoro-2'-deoxy-                                                              uridine                                                                       Compounds for                                                                 comparison:                                                                   5-fluoro-2'-deoxy-                                                                        200     400      54      2.0                                      uridine                                                                       3',5'-dihexanoyl-5-                                                                       10       40      38      4.3                                      fluoro-2'-deoxy-                                                              uridine                                                                       3',5'-dipalmitoyl-5-                                                                      0.8     4.0      55      5.0                                      fluoro-2'-deoxy-                                                              uridine                                                                       ______________________________________                                    

As seen from Table 1, the compounds of this invention display a strongantitumor effect in very small doses and also have a very goodtherapeutic index.

EXAMPLE 9

Antitumor activity of 5-fluoro-2'-deoxyuridine derivatives (in oraladministration)

The antitumor effect of 3',5'-didecanoyl-5-fluoro-2'-deoxyuridine,3',5'-didodecanoyl-5-fluoro-2'-deoxyuridine and3',5'-ditetradecanoyl-5-fluoro-2-deoxyuridine selected from thecompounds of this invention on mouse leukemia L1210 was examined ascompared with their parent compound 5-fluoro-2'-deoxyuridine and alsowith 3',5'-diotanoyl-5-fluoro-2'-deoxyuridine.

BDF, mice (male, 6-week-oil, Ca 24 g in groups of 5 mice), which wereabdominally implanted with 1×10⁵ cells of mouse leukemia L1210, whichhad been collected after 7-day cultivation in other mice, were used asexperimental animals.

The respective agents were administered orally three times, i.e. on thefirst day, third day, and fifth day, starting 24 hours after theimplantation of tumor cells.

The antitumor effect of the respective agents were indicated by the rateof increase in the length of the survival period obtained from thegroups given the agents against the length of the survival period of thecontrol group (not given the agents).

The results are shown in Table 2. As seen from Table 2,3',5'-didecanoyl-5-fluoro-2'-deoxyuridine,3',5'-didodecanoyl-5-fluoro-2'-deoxyuridine, and3',5'-ditetradecanoyl-5-fluoro-2'-deoxyuridine which are included in thecompounds of this invention showed much higher antitumor effect also inoral administration as compared with their parent compound,5-fluoro-2'-deoxyuridine, and3',5'-2-dioctanoyl-5-fluoro-2'-deoxyuridine.

                  TABLE 2                                                         ______________________________________                                                       Dose             Change in                                                    (mg/     ILS     body weight                                   Compound       kg/day)  (%)     (1-4 d, g/mouse)                              ______________________________________                                        Compounds of                                                                  this invention:                                                               3',5'-didecanoyl-5-fluoro-                                                                    10       7      -0.8                                          2'-deoxyuridine                                                                               30      26      -1.4                                                         100      15      -2.4                                                         300      20      -3.8                                          3',5'-didodecanoyl-5-                                                                         10       0      +1.0                                          fluoro-2'-deoxyuridine                                                                        30       5      +1.0                                                         100      15       0                                                           300      40      -2.6                                          3',5'-tetradecanoyl-5-                                                                        1        3      +2.2                                          fluoro-2'-deoxyuridine                                                                        3        3      +2.0                                                          10      27      -0.8                                                          30      33      -1.4                                                         100      17      -2.4                                          Compounds                                                                     for comparison:                                                               5-fluoro-2'-deoxyuridine                                                                      10      15      +0.6                                                          30      10      +1.8                                                         100      15      -4.2                                                         300      10      -5.2                                          3',5'-dioctanoyl-5-fluoro-                                                                    10      -5      +1.8                                          2'-deoxyuridine                                                                               30       3      +1.6                                                         100      -5      -2.8                                                         300       5      -2.2                                          Control        --        0      +0.4                                          ______________________________________                                    

EXAMPLE 10

Rate of hydrolysis of 5-fluoro-2'-deoxyuridine derivatives by esterase

Experiments were made on the 5-fluoro-2'-deoxyuridine derivative of thisinvention to determine the rates of releasing its parent compount(5-fluoro-2'-deoxyuridine) in the enzymatic hydrolysis conducted by useof estrase extracted from the porcine liver.

A 10 μg/ml isotonic solution of the experimental compound in a phosphatebuffer (pH 7.00) was prepared and the estrase (manufactured by Sigma)extracted from the porcine liver was added to the solution at 37° C. insuch a way as to provide the enzyme concentration ranging from 0.03units/ml to 150 units/ml. Then the sample (10 μl) was injected into anHPLC column periodically to determine the amount of5-fluoro-2'-deoxyuridine release by the enzyme reaction.

The time (t1/2) required for 1/2 the amount of the5-fluoro-2'-deoxyuridine derivative, which was used in the hydrolysis,to be converted into its parent compound at the respectiveconcentrations of the enzyme is shown as the index to indicate the rateof hydrolysis.

The results are as shown in Table 3.

                  TABLE 3                                                         ______________________________________                                                              Enzyme                                                                        concentration                                                                            t 1/2                                        Compound              (units/ml) (min)                                        ______________________________________                                        Compounds of this invention:                                                  3',5'-didecanoyl-5-fluoro-2'-deoxyuridine                                                           0.3        140                                                                0.6        72                                           3',5'-didodecanoyl-5-fluoro-2'-deoxyuridine                                                         150.0      4000                                         3',5'-diadipoyl-5-fluoro-2'-deoxyuridine                                                            6.0        438.7                                                              12.0       216.5                                        3',5'-diglutaryl-5-fluoro-2'-deoxyuridine                                                           150.0      437.2                                        3',5'-disuccinyl-5-fluoro-2'-deoxyuridine                                                           150.0      3224.0                                       Compounds for comparison:                                                     3',5'-dipropanoyl-5-fluoro-2'-deoxyuridine                                                          3.0        47.4                                                               6.0        24.2                                         3',5'-dibutyryl-5-fluoro-2'-deoxyuridine                                                            0.75       42.4                                                               1.50       21.7                                         3',5'-dihexanoyl-5-fluoro-2'-deoxyuridine                                                           0.045      32.5                                                               0.075      24.0                                                               0.15       10.0                                         3',5'-dioctanoyl-5-fluoro-2'-deoxyuridine                                                           0.03       18.2                                                               0.045      15.6                                         ______________________________________                                    

As shown in Table 3, the understanding that the compounds of thisinvention release their parent compound (5-fluoro-2'-deoxyuridine) at avery slow rate in the enzymatic reaction and that they have a propertyto slowly release 5-fluoro-2'-deoxyuridine in the enzyme system in vivoafter their administration in the living body can be well supported.

EXAMPLE 11

Behavior to release FUdR from the compound due to the enzyme system inthe plasma

The release rates of 5-fluoro-2'-deoxyuridine (FUdR) from the compoundof this invention at 37° C. in the rat plasma (20%), diluted with 0.1Mphosphate buffer, pH 7.0, were measured.

The compound of this invention was added to the plasma diluted with anisotonic solution of phosphate buffer to a concentration of 4×10⁻⁵ M(corresponding to FUdR 9.85 μg/ml) and was incubated at 37° C. Thesample (10 μl) was injected into an HPLC column periodically todetermine the amount (μg/ml) of 5-fluoro-2'-deoxyuridine released by theenzymatic reaction.

The results are as shown in Table 4.

As seen from Table 4, it is clear that the compounds of this inventionrelease their parent compound at a very slow rate in the rat plasma ascompared with 3',5'-dihexanoyl-5-fluoro-2'-deoxyuridine and3',5'-dioctanoyl-5-fluoro-2'-deoxyuridine and that they have a propertyto release 5-fluoro-2'-deoxyuridine slowly in vivo after theiradministration into the living body.

                  TABLE 4                                                         ______________________________________                                                      Release rate of 5-fluoro-2'-                                                  deoxyuridine (μg/ml)                                                         after  after  after after                                                                              after                                                100    200    300   400  500                                  Compound        min    min    min   min  min                                  ______________________________________                                        Compounds of this invention:                                                  3',5'-didecanoyl-                                                                             4.7    6.7    7.8   8.5  9.3                                  5-fluoro-2'-deoxyuridine                                                      3',5'-didodecanoyl-                                                                           1.2    2.2    2.9   3.8  4.2                                  5-fluoro-2'-deoxyuridine                                                      Compounds for comparison:                                                     3',5'-dihexanoyl-                                                                             7.8     9.85  --    --   --                                   5-fluoro-2'-deoxyuridine                                                      3',5'-dioctanoyl-                                                                              9.85  --     --    --   --                                   5-fluoro-2'-deoxyuridine                                                      ______________________________________                                    

EXAMPLE 12

Preparation of injections

The compound (3',5'-dimyristoyl-5-fluoro-2'-deoxyuridine) of thisinvention and 0.5-1% polyoxyethylene hardened castor oil were dissolvedin an aqueous solution (pH 6.00-7.50) to obtain an injection containing0.3 mg-1 mg/ml of the compound.

EXAMPLE 13

Preparation of tables

Tablets of the following composition were prepared according to theordinary method of forming tablets.

    ______________________________________                                        Compound of this invention                                                    (3',5'-didodecanoyl-5-fluoro-2'-deoxyuridine)                                                          50 mg                                                Lactose                  50 mg                                                Corn starch              40 mg                                                Calcium carboxymethyl cellulose                                                                        57 mg                                                Magnesium stearate        3 mg                                                Total                    200 mg                                               ______________________________________                                    

EXAMPLE 14

Preparation of capsules

Hard gelatin capsules having the following composition per capsule wereprepared according to the ordinary method of filling capsules.

    ______________________________________                                        Compound of this invention                                                    (3',5'-ditetradecanoyl-5-fluoro-2'-deoxyuridine)                                                        10 mg                                               Lactose                   120 mg                                              Crystalline cellulose     67 mg                                               Magnesium stearate         3 mg                                               Total                     200 mg                                              ______________________________________                                    

EXAMPLE 15

Preparation of liposomes

Liposomes preparation were obtained according to the ordinary methodunder ultrasonic treatment by use of an isotonic sodium chloridesolution containing 26 mg of lecithin, 5 mg of cholesterol, and 40 mg ofa compound (3',5'-didodecanoyl-5-fluoro-2'-deoxyuridine) of thisinvention.

Industrial Applications

The 5-fluoro-2'-deoxyuridine derivatives of this invention exhibit theantitumor effect of high level in low dose, provide an extremelyremarkable safety, and have an excellent property of sustained releasingeffect in vivo of 5-fluoro-2'-deoxyuridine. They are accordingly veryuseful as the remedy for malignant tumors.

We claim:
 1. The 5-fluoro-2'-deoxyuridine derivative expressed by thefollowing formula (I') ##STR7## wherein R₁ ' and R₂ ' are the same ordifferent from each other, each representing an alkyl group of 1 to 18carbon atoms having a carboxyl group as substituent, or itspharmacalogically acceptable salt.